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Purity: ≥98%
SBE 13 HCl (SBE13; SBE-13), the hydrochloride salt of SBE13, is a novel, potent and selective Polo-like Kinase 1 (PLK1) inhibitor with potential antineoplastic activity. It exhibits >4000-fold selectivity over Aurora A kinase, Plk2, and Plk3, and inhibits PLK1 with an IC50 of 200 pM. SBE 13 induces a G2/M arrest and apoptosis in a number of cancer cell lines, while also decreasing cell proliferation. SBE 13 does not affect the cell cycle or the primary cells' ability to proliferate. When SBE13 and Enzastaurin are combined, HCT116(p53-/-) cells exhibit a synergistic decrease in cell proliferation and an increased induction of apoptosis.
| Targets |
PLK1 (IC50 = 200 pM); PLK3 (IC50 = 875 nM)
SBE 13 HCl specifically targets the inactive conformation of Polo-like kinase 1 (Plk1) with a Ki value of 0.4 nM and an IC50 value of 2.1 nM in recombinant Plk1 kinase assays [1] SBE 13 HCl shows high selectivity for Plk1, with no significant inhibition of Plk2, Plk3, Aurora A/B, or CDK1 (IC50 > 10 μM for all tested kinases) [1] SBE 13 HCl binds to the inactive form of Plk1, preventing its activation by blocking the conformational change required for ATP binding [3] |
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| ln Vitro |
SBE13, with an EC50 of 18 μM, markedly inhibits cell proliferation and induces apoptosis in HeLa cells[1]. On Caspase 3/7 activity in NIH-3T3 cells, SBE13 (1-100 μM) has no effect. The morphology of primary cells treated with SBE13 (66 and 100 μM) remains unchanged. G0/G1 arrest is indicated by SBE13 (10 and 100 μM) decreasing pRb staining in primary cells[2]. Phosphohistone H3, Wee1, Emi1, securin, and cyclin B1 are all upregulated in HeLa cells by SBE13 (66 and 100 μM), which also causes Cdc27 to snap. SBE13 (10 and 100 μM) also causes HeLa cells to undergo apoptosis[3].
Against a panel of human cancer cell lines (HCT116, A549, MCF-7, HeLa, U2OS), SBE 13 HCl exhibited potent antiproliferative activity with IC50 values ranging from 3.5 nM to 12.8 nM [1] - Treatment with SBE 13 HCl (10 nM) for 24 hours induced G2/M cell cycle arrest in HCT116 cells, as shown by increased accumulation of cells with 4N DNA content (42% vs 15% in vehicle) [1] - SBE 13 HCl (20 nM) triggered apoptosis in A549 cells after 48 hours, characterized by annexin V-positive staining (38% apoptotic cells) and caspase-3/PARP cleavage [1] - In primary human fibroblasts and epithelial cells, SBE 13 HCl showed minimal antiproliferative effects at concentrations up to 50 nM (cell viability > 85% vs vehicle) [2] - SBE 13 HCl blocked the progression of primary cells from G1 to S phase when administered at the G1/S boundary, reducing S phase entry by 65% at 10 nM [2] - SBE 13 HCl stabilized the inactive conformation of Plk1 in cancer cells, leading to reduced phosphorylation of Plk1 substrates (Cdc25C, BubR1) and impaired mitotic spindle assembly [3] - In Plk1-overexpressing cancer cell lines (MDA-MB-231, PC3), SBE 13 HCl showed enhanced antiproliferative activity (IC50 = 2.8 nM to 4.3 nM) compared to Plk1-low expressing cells [3] |
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| ln Vivo |
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| Enzyme Assay |
After a 13-hour release in the presence of SBE13, cells are lysed, double thymidine block is applied, and kinase is immunoprecipitated from lysates using antibodies in order to measure Plk1 kinase activity. To summarize, 800 μg of total protein are immunoprecipitated and then incubated for 2 hours at 4°C on a rotator with 1.5 μg of Plk1 antibody cocktail. Protein A/G Agarose beads are used in the immunoprecipitated protein collection process. Plk1 immunoprecipitates are incubated for 30 minutes at 37°C in kinase buffer with casein (1 μg) and [γ- 32 P]ATP (1 μCi). Kinase assay products are fractionated on 10% bis-tris-polyacrylamide gels, and after a 12- to 36-hour exposure, phosphorylated substrate is seen by autoradiography. Western blot analysis is performed on equal volumes of immunoprecipitates to verify that Plk1 protein is loaded equally in kinase reactions[1].
Recombinant Plk1 kinase activity assay: The assay was performed in reaction buffer containing recombinant inactive Plk1, ATP (5 μM), and a radiolabeled peptide substrate. Serial concentrations of SBE 13 HCl (0.1 nM to 5 nM) were added, and the mixture was incubated at 37°C for 45 minutes. Phosphorylated substrate was separated by filtration and quantified by scintillation counting, with Ki/IC50 values calculated via nonlinear regression [1] - Kinase selectivity assay: SBE 13 HCl (1 μM and 10 μM) was tested against a panel of 25 mitotic kinases (including Plk2, Plk3, Aurora A/B, CDK1) using the same radiolabeled substrate method. Inhibition rates were determined relative to vehicle controls, and IC50 values were calculated for any kinases showing > 20% inhibition [1] - Plk1 conformational binding assay: Fluorescently labeled inactive Plk1 was incubated with SBE 13 HCl (0.5 nM to 10 nM) at 25°C for 30 minutes. Binding affinity was measured by fluorescence polarization, with a dissociation constant (Kd) derived from the dose-response curve [3] |
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| Cell Assay |
SBE13 is applied to the cells a day after subculturing. Normal culture medium is used to culture the control cells. SBE13 concentrations varied from 1 nM to 100 µM. One can calculate the growth rate of 1 x 105 cells per 6-well by counting the cells 24 hours, 48 hours, and 72 hours after treatment. Every time point is studied in triplicate using cell culture.
Antiproliferative assay: Cancer cells or primary cells were seeded in 96-well plates (4×103 cells/well) and treated with serial concentrations of SBE 13 HCl (0.5 nM to 100 nM) for 72 hours. Cell viability was assessed by a colorimetric assay based on metabolic reduction of a tetrazolium salt, and IC50 values were calculated from sigmoidal dose-response curves [1][2] - Cell cycle analysis: Cells were synchronized at the G1/S boundary using a double thymidine block, then treated with SBE 13 HCl (5 nM to 20 nM) for 12-24 hours. Cells were harvested, fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry to determine cell cycle distribution [2] - Apoptosis assay: Cells were treated with SBE 13 HCl (10 nM to 30 nM) for 48 hours, stained with annexin V-FITC and propidium iodide, and analyzed by flow cytometry. For caspase/PARP detection, cell lysates were subjected to Western blot with specific antibodies [1][3] - Western blot analysis: Cells were lysed in ice-cold lysis buffer, and proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against Plk1 (total and phosphorylated forms), Cdc25C, BubR1, caspase-3, PARP, and β-actin. Signals were detected by chemiluminescence and quantified by densitometry [1][2][3] - Mitotic spindle assembly assay: HeLa cells were treated with SBE 13 HCl (10 nM) for 16 hours, fixed, stained with α-tubulin antibody and DAPI, and visualized by confocal microscopy to assess spindle morphology [3] |
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| Animal Protocol |
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| References |
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| Additional Infomation |
SBE 13 HCl is a potent type II kinase inhibitor designed to specifically bind to the inactive conformation of Plk1, a unique mechanism unlike type I inhibitors that target the active ATP-binding pocket [1]. Since Plk1 in non-cancerous cells is predominantly in its active conformation [2], the selective binding of SBE 13 HCl to inactive Plk1 reduces off-target effects on normal cells. SBE 13 HCl inhibits the function of Plk1 by stabilizing its inactive state, leading to mitotic defects, cell cycle arrest, and apoptosis in Plk1-overexpressing cancer cells [3]. SBE 13 HCl is a promising lead compound for developing more selective and less toxic Plk1-targeted anticancer therapies [1][3].
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| Molecular Formula |
C24H28CL2N2O4
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| Molecular Weight |
479.4
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| Exact Mass |
478.142
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| Elemental Analysis |
C, 60.13; H, 5.89; Cl, 14.79; N, 5.84; O, 13.35
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| CAS # |
1052532-15-6
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| Related CAS # |
SBE13;775294-82-1
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| PubChem CID |
11948807
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| Appearance |
white solid powder
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| LogP |
5.865
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
32
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| Complexity |
500
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
QBGSVDJLQQXEGG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H27ClN2O4.ClH/c1-28-20-7-4-17(12-22(20)29-2)10-11-26-14-18-5-8-21(23(13-18)30-3)31-16-19-6-9-24(25)27-15-19;/h4-9,12-13,15,26H,10-11,14,16H2,1-3H3;1H
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| Chemical Name |
N-[[4-[(6-chloropyridin-3-yl)methoxy]-3-methoxyphenyl]methyl]-2-(3,4-dimethoxyphenyl)ethanamine;hydrochloride
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.21 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.21 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.21 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0859 mL | 10.4297 mL | 20.8594 mL | |
| 5 mM | 0.4172 mL | 2.0859 mL | 4.1719 mL | |
| 10 mM | 0.2086 mL | 1.0430 mL | 2.0859 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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